Human TGF-beta has been isolated from human blood platelets and placenta and purified to essential homogeneity using sequential gel filtration cation-exchange chromatography and high performance liquid chromatography (HPLC). The purified protein was characterized as having a molecular weight of 25,000 daltons, composed of 2 sub-units of 12,500 daltons each held together by disulfide bonds. Its molecular weight, sub-unit structure and amino acid composition differed from those of platelet derived growth factor. See European Patent Application EP 128849 (Dec. 19, 1984), priority applications U.S. Ser. No. 500,832 (Jun. 3, 1983) and U.S. Ser. No. 500,927 (Jun. 3, 1983).
An alternative method for purifying TGF-beta from platelets or conditioned media utilizes acid-ethanol extraction, cation-exchange separation on the extract, carrying out hydrophobic separations on the active fractions so as to obtain a homogeneous preparation and is described in European Patent Application 322842-A (Jul. 12, 1989). The purified product is indicated to be useful in wound healing and tissue repair.
Production of TGF-beta 1 utilizing recombinant DNA techniques is described in JP61219395 (Sep. 29, 1986) having priority from U.S. patent application Ser. No. 715,142 (Mar. 22, 1985) assigned to Genentech as well as in JP63028386 (Feb. 6, 1988) assigned to Asahi Chemical Industry Co. Ltd. Both disclosures insert the cloned human gene coding for TGF-beta 1 into eukaryotic cell lines for expression. The protein product was indicated to be useful in promoting anchorage-dependent and independent growth in cell culture.
Further suggestion as to the use of TGF-beta in wound healing is found in PCT application WO 8911293-A (Nov. 30, 1989) based on U.S. patent application Ser. No. 196,975 (May 20, 1988) where a composition containing both purified insulin-like growth factor-1 and purified TGF-beta in ratios of 1:4 to 25:1 are employed.
Polyunsaturated fatty acids have been known to exhibit cytotoxicity against tumor cells based on a proposed ability to stimulate the production of superoxide radicals. Thus Begin et al., Journal of the National Cancer Institute, 80, No. 3, 188-194 (Apr. 6, 1988) tested a series of fatty acids containing 2, 4, 5, and 6 double bonds, which were compared to gamma-linolenate (3 double bonds). The results showed that effectiveness of fatty acid induced cytotoxicity on cancer cells correlated with intracellular thiobarbituric acid-reactive material content. Gamma-linolenate and arachidonate with 3 and 4 double bonds gave the highest cytotoxicity and highest correlation. All of the experiments described in this study were carried out in the presence of serum, which contains TGF-.beta.1. Earlier results to similar effect were reported by the same group in a publication in the Journal of the National Cancer Institute, 77, No. 5 at 1054-1062 (November, 1986). This study also demonstrated that polyunsaturated fatty acid-induced cytotoxicity was relatively specific for tumor cells. Concentrations of fatty acids which killed tumor cells had little or no effect on the growth of nontransformed cells.